Evaluation of the efficacy of two postweaning colibacillosis vaccines in a field herd with PRRS circulation during postweaning stage.

Postweaning diarrhea (PWD) and PRRS are two major concerns in swine production, which association has not been consistently explored. In the current scenario of restrictions in the use of antibiotics and ZnO, vaccination is more relevant to control PWD, but PRRS virus circulation may compromise the immune protection conferred by postweaning colibacillosis vaccines. We evaluated the efficacy of two postweaning colibacillosis vaccines (parenteral and oral) in a commercial herd affected by an outbreak of PWD and with PRRS circulation in postweaning. Five groups were studied during the postweaning period: one control (Group 1) and four vaccinated: two with each postweaning colibacillosis vaccine administered alone (Groups 2 and 3) or with sow vaccination against PRRS (Groups 4 and 5). We evaluated the effects on piglet weight, average daily weight gain and in the percentage of piglets with diarrhea, its duration, lethality and mortality. PRRS viremia and anti-PRRS antibodies were evaluated by qPCR and ELISA. Regarding control group, colibacillosis vaccination generally improved most of the measured parameters; but significant improvements were only observed in Groups 4 and 5 (p < 0.05). Moreover, cases of diarrhea occurred at different ages: in Groups 2 and 3 the peak of cases occurred just after ZnO was removed from the feed compared to Group 1, while in Groups 4 and 5 no peak was observed. This suggests that postweaning colibacillosis vaccination may be compromised by the PRRS circulation. In PRRS endemic herds an effective protection against PWD through vaccination may require PRRS vaccination to obtain a better performance.

Molecular detection of Rickettsia spp. in wild ungulates and their ticks in Mediterranean areas of southwestern Spain.

Wildlife is an important reservoir of zoonotic pathogens. The objective of the present study was to assess the importance of wild ungulates in the epidemiology of Rickettsia spp. Ticks and spleen samples were collected from 262 red deer (Cervus elaphus) and 83 wild boar (Sus scrofa) hunted in southwestern Spain over a 5-year period. DNA was extracted from tick pools (n = 191) and spleens (n = 345), and two nested PCR assays targeting the rOmpA and rOmpB genes were used to detect Rickettsia DNA. Five tick species were identified (Hyalomma lusitanicum, Dermacentor marginatus, Ixodes ricinus, Rhipicephalus bursa and Haemaphysalis sulcata). Rickettsia DNA was detected in 31 (16.2%) tick pools and two red deer spleen samples (0.8%). Four validated Rickettsia species (R. slovaca, R. monacensis, R. helvetica and R. raoultii), one uncultivated species (Candidatus R. rioja) and two uncharacterized Rickettsia spp. were detected in ticks. R. helvetica and R. slovaca were also detected in spleen samples from red deer. The overall prevalence in ungulate spleen samples was lower than in tick pools suggesting that these ungulates do not play a major role in the transmission of Rickettsia spp. However, their importance as spreaders of positive ticks cannot be ruled out. The results present a challenge for the veterinary and public health communities since most of the Rickettsia spp. detected are pathogenic. Furthermore, the new Rickettsia species could be potential pathogens. For these reasons, identifying Rickettsia species present in ticks and wildlife is of particular interest to clarify their sylvatic cycle and establish appropriate control measures.

Cephenemyia stimulator (Diptera: Oestridae) myiasis in a roe deer (Capreolus capreolus) from Portugal.

Herein we present a nasopharyngeal myiasis case by Cephenemyia stimulator in a roe deer hunted in Trás-os-Montes (NE Portugal). Preliminary inspection showed one larva sorting through the nostrils and the examination of the nasopharyngeal cavity showed more than 15 larvae in the glottis and retropharyngeal recesses. Four larvae were collected and stored into 70% ethanol for morphological and molecular identification. Three of the larvae were identified as third instars and the other one as a prepupa of Cephenemyia stimulator, being the first confirmation of this species in roe deer from Portugal. C. stimulator is currently widely distributed in roe deer from central and northern Spain so, the transboundary natural dispersal of these cervids would explain the introduction of this myiasis in Portugal. Further studies are needed to monitor the spreading of this infection in the westernmost populations of European roe deer.

Pedicle myiasis by Lucilia caesar (Diptera, Calliphoridae): An emerging disease in roe deer from north-western Spain.

A total of 35 male roe deer (Capreolus capreolus) from Lugo province (north-western Spain) were examined for pedicle myiasis between 2020 and 2022. All these animals had died by different causes and were examined or preserved just after their death to avoid post-mortem infestations. After external and internal head inspection, five animals were diagnosed with severe myiasis at the basis of the antlers (14.29%; 95% confidence interval = 5.38-31.04). All the affected bucks presented a cutaneous wound (1.5-15 cm in diameter) around the pedicles with extensive tissue destruction, exposition of frontal and parietal bones, and massive infestation by dipteran larvae (2-12 mm long). Four of five roe deer showed whitish egg clusters adhered to the basis of the antlers. Moreover, in one animal, six larvae had penetrated the cranial cavity, and in two of them larvae were also found in the nasopharyngeal cavity. All the cases were recorded in summer (May-August). Morphological identification and subsequent molecular confirmation revealed that all animals were infested by different larval stages of Lucilia caesar (Diptera: Calliphoridae). This study represents the first report of pedicle myiasis in Spain. Since this disease is reported sporadically, the detection of five cases in a short period of time suggests an increase in the incidence of this myiasis.

Cardiorespiratory nematodes and co-infections with gastrointestinal parasites in new arrivals at dog and cat shelters in north-western Spain.

Metastrongyloid and trichuroid cardiorespiratory nematodes of dogs and cats are considered emergent in several European countries, and an increase in the number and extent of endemic foci has been described. Since data on their prevalence are limited in this continent, faecal samples from new arrivals (365 dogs and 65 cats) at two animal shelters in North-western Spain were analysed using both floatation and Baermann techniques. In order to confirm the microscopic identification of Metastrongylidae first stage larvae, molecular characterization based on the sequence of the ITS-2 was performed. The possible influence of some variables such as the species, sex and age of the animals and the co-infection with other gastrointestinal parasites on the prevalence of cardiorespiratory nematodes was analysed. The most prevalent metastrongylid was Aelurostrongylus abstrusus (15.4%) followed by Angiostrongylus vasorum (4.1%) and Crenosoma vulpis (1.1%). Regarding trichuroids, Eucoleus aerophilus and/or Eucoleus boehmi eggs were detected in 28 dogs (7.7%) and four cats (6.2%). Almost all animals positive to cardiorespiratory nematodes (86.8%) were co-infected with gastrointestinal parasites. The prevalence of Metastrongylidae and respiratory trichuroids was significantly higher in dogs co-infected with Taenia spp. and Toxocara canis or Giardia duodenalis and Sarcocystis spp., respectively. In cats, a significant higher prevalence of Metastrongylidae nematodes was found in animals co-infected with Toxocara cati. Our results reveal that cardiorespiratory nematodes are common in companion animals from north-western Spain, showing higher prevalences than those previously reported from this country. This investigation represents the first report of C. vulpis, E. aerophilus and E. boehmi in dogs from Spain. The identification of a number of zoonotic parasites is of public health concern. Our results indicate that these nematodes should be included in the differential diagnosis of dogs and cats from north-western Spain showing respiratory or cardiac clinical signs.

Antimicrobial Susceptibility of Enterotoxigenic Escherichia coli from Diarrhoeic Neonatal Calves in Spain.

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens involved in neonatal calf diarrhoea (NCD) causing high economic losses in dairy farms. Antibiotic treatment is common in cases of systemic illness caused by NCD, but antimicrobial susceptibility tests (AST) are usually not performed. Thus, the aim of this study was to characterize the antimicrobial susceptibility of ETEC strains obtained from calves with diarrhoea between 2018-2020. Faecal samples (n = 420) were analyzed to detect the typical ETEC virulence factors F5 and STa. Positive samples were cultured to identify and isolate ETEC strains (n = 41) and ASTs were performed. Our results are alarming since ETEC strains resistant to three or more families of antimicrobials were detected in all isolates. Only four antibiotics (ceftiofur, cefoperazone, cefquinome and gentamicin) presented efficacy against more than 90% of the ETEC strains, while the other ten antibiotics were effective against less than 40% of the strains. In addition, a high number of strains were resistant to most first-line antimicrobials used in veterinary practice. For this reason, when ETEC infection is suspected, an AST must always be performed to select the most appropriate antimicrobial in each case and to avoid the emergence of new resistance mechanisms.

Mixed Nasopharyngeal Myiasis by Bots and Blowflies in a Roe Deer (Capreolus capreolus).

A mixed nasopharyngeal infestation by larvae of Cephenemyia stimulator (Diptera: Oestridae) and Lucilia caesar (Diptera: Calliphoridae) was detected in a roe deer (Capreolus capreolus) killed in a car collision in Lugo, Spain, in July 2020. This article stresses the importance of correctly identifying larvae in myiasis cases.

Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

An Update on Cephenemyiosis in the European Roe Deer: Emergent Myiasis in Spain.

Cephenemyia stimulator is a Palearctic species developing in the nasal cavity and pharynx of roe deer (Capreolus capreolus). It is widely spread in the range of distribution of this ungulate in Europe. Since the first report of C. stimulator in Spain in 2001, a rapid geographic expansion has been observed, first in the north of the country, with high prevalence and intensities of infestation that caused some mortal cases, and, lately, also in Extremadura and Andalucía, the southernmost populations of European roe deer. These observations suggest an adaptation of this parasite to different ecosystems of the Iberian Peninsula. Almost simultaneously, C. stimulator is also expanding its range to northern Europe, with the first cases being reported in Sweden. Thus, Cephenemyia stimulator may be an example of a parasite currently displaying distributional changes along its southernmost and northernmost range margins. Thus, it is of the utmost importance to unravel all the epidemiological and clinical aspects of this myiasis, as well as implementing surveillance measures including reliable and non-invasive diagnostic techniques to monitor its expansion and adaptation to different ecosystems and/or hosts and to reduce the negative impact on roe deer populations.

The Goat as a Risk Factor for Parasitic Infections in Ovine Flocks.

Small ruminants in northwestern Spain are frequently managed in mixed flocks. Sheep-goat joint management has not been considered a risk factor for parasite infection, so the main objective of this study was to establish if mixed management with goats supposes a risk factor for parasitic infections in ovine flocks. Two thousand and ninety-three sheep were sampled from 74 commercial meat ovine flocks for diagnostic of the main parasites. Goat contact was a risk factor for sheep to be infected by protostrongylids, Dictyocaulus filaria, gastrointestinal nematodes and Eimeria spp. In contrast, Moniezia, Fasciola hepatica, Dicrocoelium dendriticum and Paramphistomidae prevalences were not influenced. Sheep-goat mixed management can be considered as a risk factor, since goats would act as a source of pasture contamination for interspecific parasites (protostrongylids, Dictyocaulus filaria and gastrointestinal nematodes). In relation to host-specific parasites, such as Eimeria spp., goats cannot be considered as a source for sheep, but competition for food and spaces between both small ungulates can suppose a reduced grazing area to sheep, provoking high environmental contamination and stress that facilitate their infection. Future epidemiological studies for parasitic infections in small ruminants should consider sheep-goat mixed management as a possible risk factor to be included in multivariate analyses.

Analysis of environmental dust in goat and sheep farms to assess Coxiella burnetii infection in a Q fever endemic area: Geographical distribution, relationship with human cases and genotypes.

Real-time PCR analysis of environmental samples (dust and aerosols) is an easy tool to investigate the presence of Coxiella burnetii in the farm environment. The aim of this study was to assess the distribution of C. burnetii DNA in dust collected inside animal premises from 272 small ruminant farms in Bizkaia (northern Spain), a region with recent reports of human Q fever cases and outbreaks. Within each farm, 5 samples of dust were collected from difference surfaces, and data on animal census, management procedures, characteristics of the premises and geographic location were collected. Real-time PCR analysis of the dust samples detected presence of C. burnetii DNA in 98 farms (36.0%), flock-prevalence being higher in sheep (38.9%) or mixed ovine-caprine production systems (36.8%), compared to goats (25.0%). Larger bacterial burdens were observed in mixed farms, compared to sheep (p < .05). Single nucleotide polymorphism (SNP) analysis identified 5 different genotypes, with SNP8 being the predominant genotype (73%), followed by SNP6 (11%), SNP2 (9%), SNP4 (5%) and SNP1 (2%). Proportion of farms where C. burnetii DNA was detected differed among the different agricultural counties, and a higher proportion of C. burnetii DNA positive farms was associated with the occurrence of recent human Q fever outbreaks at several geographical locations. Dust sampling in domestic ruminant farms coupled with real-time PCR to screen for the presence of C. burnetii and estimate bacterial load can be a useful tool to identify herds and regions with high prevalence, define priority actions and monitor the effect of control measures. If combined with molecular genotyping and spatial distribution maps, it can help to identify farm contamination sources and trace the origin of human outbreaks.

One Health Approach: An Overview of Q Fever in Livestock, Wildlife and Humans in Asturias (Northwestern Spain).

This study aimed to investigate the seroprevalence of C. burnetii in domestic ruminants, wild ungulates, as well as the current situation of Q fever in humans in a small region in northwestern Spain where a close contact at the wildlife-livestock-human interface exists, and information on C. burnetii infection is scarce. Seroprevalence of C. burnetii was 8.4% in sheep, 18.4% in cattle, and 24.4% in goats. Real-time PCR analysis of environmental samples collected in 25 livestock farms detected Coxiella DNA in dust and/or aerosols collected in 20 of them. Analysis of sera from 327 wild ungulates revealed lower seroprevalence than that found in domestic ruminants, with 8.4% of Iberian red deer, 7.3% chamois, 6.9% fallow deer, 5.5% European wild boar and 3.5% of roe deer harboring antibodies to C. burnetii. Exposure to the pathogen in humans was determined by IFAT analysis of 1312 blood samples collected from patients admitted at healthcare centers with Q fever compatible symptoms, such as fever and/or pneumonia. Results showed that 15.9% of the patients had IFAT titers ≥ 1/128 suggestive of probable acute infection. This study is an example of a One Health approach with medical and veterinary institutions involved in investigating zoonotic diseases.

Efficacy of Two Commercial Ready-To-Use PCV2 and Mycoplasma hyopneumoniae Vaccines under Field Conditions.

Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are economically important pathogens in swine farms. Vaccination is the main preventive measure for both infections. In order to test two ready-to-use bivalent vaccines, 646 piglets from a herd actively infected with both pathogens were stratified according to the sow parity number and randomly assigned to three groups: A and B were vaccinated with two different vaccines, respectively, while C remained as the unvaccinated control. Vaccine efficacy was assessed based on the weight, average daily weight gain (ADWG), degree of lung lesions, presence of PCV2 viremia by qPCR and presence of PCV2 and M. hyopneumoniae antibody levels by ELISA. Our data revealed that the sow parity did not influence the vaccine outcomes. Good results for most of the analyzed parameters were observed in both vaccinated groups. ADGW and final weight were higher and lung lesions were less evident in both vaccinated groups than in the control one, but only Group A showed a significant improvement. PCV2 viremia was not detected in Group A, but it did appear in Group B coinciding with its peak in Group C. Finally, both the PCV2 and M. hyopneumoniae serological patterns differed depending on the employed vaccine.

Las medidas gubernativas en la crisis sanitaria de 1803-1805 / Governamental measures in the health crisis of 1803-1805

La crisis de producción de 1803-1805 fue acompañada de muchos casos de fiebres palúdicas agravadas por la pobreza del mundo rural. Este trabajo intenta una aproximación a las medidas de alivio de pobres y enfermos mediante el análisis de los expedientes de petición de socorro presentados al Consejo de Castilla. El gobierno actuó sobre una línea doble; por un lado la ayuda a los enfermos con provisiones de quina y el recurso a la sopa económica, y por otro la promoción de trabajos públicos para dar trabajo a los jornaleros desocupados. Los poderes locales descubren las pésimas condiciones higiénicas de sus pueblos y se emprenden obras de construcción de fuentes y drenaje de aguas estancadas. El resultado global fue muy limitado debido a la escasez de fondos disponibles porque todo el peso de la financiación recaía sobre los ayuntamientos. Al llevar a cabo las obras precisas los pueblos se empobrecían ulteriormente dado que debían recurrir a la venta de comunales o a empréstitos que más tarde habrían de reembolsar.(AU)

The production crisis of 1803-1805 ushered in many cases of malaria fevers, exacerbated by the poverty of the rural world. This paper aims at approaching the measures of relief for the poor and the sick by analysing the requests for help files addressed to the Consejo de Castilla.The government acted following a twofold line: on the one hand to relieve the sick by supplying quinine and resorting to the sopa económica, and on the other by promoting public works in order to employ the unemployed day labourers. Local authorities suddenly realized their villages’ dreadful hygienic situations and started a campaign of building fountains and draining stagnant waters. The overall result was very limited owing to the scarcity of funds available, because the whole economic burden was to lay on the village councils. Carrying out these works the villages subsequently became poorer because they had to resort to selling community property or to loans that would have been repaid later.(AU)

The Age-Related Cryptosporidium Species Distribution in Asymptomatic Cattle from North-Western SPAIN.

An age-related distribution of Cryptosporidium species has been reported in cattle, with C. parvum being predominant in suckling calves, C. bovis and C. ryanae being predominant in post-weaned calves and C. andersoni being predominant in adults. However, variants to this pattern have recently been reported. Thus, fecal samples (n = 594) from asymptomatic cattle were collected in north-western Spain. Animals were classified as <1 month (G1), 1-2 months (G2), 2-12 months (G3), 12-24 months (G4) and >2 years (G5). Cryptosporidium detection and species identification were performed by SSU rRNA PCR. Individual Cryptosporidium prevalence was 16.7%; it significantly decreased with age. Cryptosporidium parvum was predominant in G1 and C. bovis was predominant in the rest of the age classes; C. bovis and C. ryanae were especially prevalent in G2 and G3. Cryptosporidium occultus was not found in suckling calves. Finally, C. andersoni and C. xiaoi were occasionally detected in G5. The presence of C. parvum in all age classes implies significant animal and public health concerns. The predominance of C. bovis in cattle older than 1 month supports the idea that the age-related pattern of Cryptosporidium species described in cattle is not fully consistent, and thus further studies are still needed to identify those factors determining the species distribution.

Epidemiological study of the association between bovine gammaherpesvirus type 4 and reproductive disease in dairy cattle from northwestern Spain.

Bovine gammaherpesvirus 4 (BoHV-4) has controversially been related with cattle reproductive disease. In the present study we analyze the relationship between exposure to BoHV-4 and reproductive performance in dairy cattle from northwestern Spain. A total of 2022 sera from 50 farms were examined to detect anti-BoHV-4 antibodies. Herd and individual reproductive records were collected to analyze association with exposure to BoHV-4. In addition, 52 abortion cases were examined to detect BoHV-4 DNA. An individual seroprevalence of 66.6 % and a herd prevalence of 98 % were found. Exposure to BoHV-4 increased with age, particularly in individuals between 26-36 months old (OR = 2.7; CI 95 %: 1.2-5.0, compared to animals < 26 months). Seroprevalence was not associated with herd fertility and herd abortion rate, but seropositive animals between 26-36 months presented prolonged calving to fertilizing insemination intervals (HR: 1.4; CI 95 %: 1.2-2.0) as well as higher odds of an unsuccessful 1st insemination (OR: 2.5; CI 95 %: 1.2-5.0). In abortion cases, BoHV-4 DNA was found in 12 vaginal swabs from 5 farms but not in any fetal tissue. Our results reveal an endemic, high and widespread exposure to BoHV-4 among dairy cattle from NW Spain with a limited impact in the reproductive performance of herds. The significantly worse reproductive performance of seropositive animals of 26-36 months of age may be the consequence of the establishment of primo-infections when moving heifers to lactation lots. Our findings may be useful to understand the potential population impact of BoHV-4.

Efficacy of Oxyclozanide and Closantel against Rumen Flukes (Paramphistomidae) in Naturally Infected Sheep.

Paramphistomosis is considered an emergent disease of ruminants in Europe. Some drugs have been found effective for treating paramphistomid infections in cattle, but data in sheep are currently limited. Thus, faecal samples from 25 adult sheep naturally infected with paramphistomids were collected weekly to test the efficacy of oxyclozanide and closantel. Three groups were performed: nine animals orally treated with a single dose of oxyclozanide (15 mg/kg bodyweight (BW) integrated the G-OXI group, whereas eight sheep orally treated with a single dose of closantel (10 mg/kg BW) were placed in a group called G-CLS. Eight untreated controls constituted the group G-CON. Oxyclozanide showed efficacies up to 90% until week 11 post-treatment, with a maximum efficacy of 98.3%, and significant differences were found between G-OXI and G-CON until the 26th week post-treatment. Closantel was insufficiently active (0-81%) throughout the study and differences compared to G-CON were never found. The present study reveals that oxyclozanide given at a single oral dose of 15 mg/kg BW is highly effective against adult rumen flukes in sheep. In addition, the use of a single oral dose of closantel at 10 mg/kg BW is not recommended for treating paramphistomid infections in sheep.

Monitoring Coxiella burnetii Infection in Naturally Infected Dairy Sheep Flocks Throughout Four Lambing Seasons and Investigation of Viable Bacteria.

Progression of Coxiella burnetii infection in four naturally infected sheep flocks, and in their farm environment, was monitored throughout four lambing seasons. Flocks with an active infection were selected based on the presence of C. burnetii DNA in bulk-tank milk (BTM) and a high seroprevalence in yearlings during the previous milking period (Spring 2015). During four consecutive lambing seasons (2015/16-2018/19), samples were collected within 1 week after each lambing period from animals (vaginal swabs, milk and feces from ewes, and yearlings) and the environment (dust indoor sheep premises). BTM samples and aerosols (outdoors and indoors) were monthly collected between lambing and the end of milking. Real-time PCR analyses showed different trends in C. burnetii shedding in the flocks, with a general progressive decrease in bacterial shedding throughout the years, interrupted in three flocks by peaks of reinfection associated with specific management practices. A significant relationship was found between C. burnetii fecal shedding and the bacterial burden detected in dust, whereas shedding by vaginal route affected the detection of C. burnetii in indoor aerosols. Three genotypes were identified: SNP8 (three flocks, 52.9% of the samples), SNP1 (two flocks, 44.8% samples), and SNP5 (one flock, two environmental samples). Coxiella burnetii viability in dust measured by culture in Vero cells was demonstrated in two of the flocks, even during the fourth lambing season. The results showed that infection can remain active for over 5 years if effective control and biosafety measures are not correctly implemented.

Prevalence and molecular characterization of Anaplasma phagocytophilum in roe deer (Capreolus capreolus) from Spain.

Anaplasma phagocytophilum can infect a wide range of vertebrates; nevertheless, some genetic variants are associated with particular species of tick vectors and animal hosts. It has been suggested that roe deer (Capreolus capreolus) mainly acts as a reservoir of several A. phagocytophilum non-pathogenic variants for other animal species. The aim of this study was to determine the prevalence and identify the genetic variants of A. phagocytophilum in roe deer from Spain in order to assess host-pathogen associations and their pathogenic potential. The spleens of 212 roe deer hunted in Spain were individually collected and analysed by a commercial qPCR kit in order to detect the presence of A. phagocytophilum DNA. Positive samples were further characterized at groESL, 16S rRNA and msp2 partial genes. The possible influence of several intrinsic (age and sex) and extrinsic factors (ecological area) on A. phagocytophilum prevalence was analysed using a logistic regression. Overall, 41.5 % of the samples resulted positive to A. phagocytophilum. The percentage of infected roe deer was significantly higher in the Mediterranean and Oceanic areas than in the Continental and Mountain regions; nevertheless, prevalence was not related to age or sex. Sequence analysis at groESL and 16S rRNA genes allowed the identification of three ecotypes (I to III) and four variants ("Y", "X", "W", "I"), respectively. A high percentage of roe deer from Spain is infected with different variants of A. phagocytophilum; these results have implications for public and animal health since some of these ecotypes and variants have been previously identified in both human and animal clinical cases.

Monitoring of the shedding and serological dynamics of Bovine gammaherpesvirus type 4 in a dairy cattle herd.

Bovine gammaherpesvirus type 4 (BoHV-4) is increasingly related with reproductive disease in cattle, but its epidemiology is not fully understood. We monitored the serological response and shedding of BoHV-4 in a positive dairy cattle farm with metritis. First, we performed an ELISA to detect BoHV-4 antibodies in all the animals (n = 104). Afterwards, ten seronegative heifers introduced in the production lot and sera samples were monthly taken for four months and then 6-10 months after introduction to detect BoHV-4 antibodies by ELISA. Moreover, a vaginal swab was taken after calving to detect BoHV-4 by PCR. Concurrently, a weekly collection of vaginal and nasal swabs and milk was performed during the first month post-partum in multiparous cows with metritis (n = 14), heifers with metritis (n = 4), heifers without metritis but positive to BoHV-4 (ELISA or PCR) (n = 2) and multiparous cows without metritis (n = 3). Seropositivity was higher in older animals and in the production lot. Three heifers which shed BoHV-4 after parturition resulted seronegative at first but eventually seroconverted. In the same vein, most heifers seroconverted after 6-10 months in the production lot (8/10). Multiparous cows shed virus by various routes: 13/14 (93 %) in vaginal secretions, 7/14 (50 %) in nasal exudates and 7/14 (50 %) in milk. However, in the other groups, shedding was only detected in vaginal swabs from the first week post-partum. Our study describes BoHV-4 shedding in field conditions. Seronegative animals may become horizontally infected when moved to a contaminated environment.